Many of the factors which participate in mammalian DNA replication are soluble in the sense that, during aqueous cell fractionation, they are recovered in the soluble cell fraction. The major part of our effort is devoted to fractionation and identification of the soluble proteins which stimulate DNA replication from 10 to the 12th power log-phase CV1-S cells. We shall independently assay for and pursue the proteins involved in RNA priming, chain tension, gap-filling and Okazaki piece joining. Extraordinary precautions are being taken to allow recombination of fractions in all possible ways and to stabilize all fractions. In additon, we intend to expand our approach to yeast cells in order to isolate the protein product of the gene cdc7, required for initiation of DNa synthesis. To help assay for initiation of DNA synthesis in mammalian cells, we are testing to see whether particular sequences are used as origins during replication of mouse cell ribosomal DNA and beta-globin (major and minor) gene sequences.